A Nonviral piggyBac Transposon-Mediated Method to Generate Large-Scale CAR-NK Cells from Human Peripheral Blood Primary NK Cells.

With the inherent antitumor function and unique "off-the-shelf" potential, genetically engineered human natural killer (NK) cells with chimeric antigen receptors (CARs) bear great promise for the treatment of multiple hematological malignancies and solid tumors. Current methods of producing large-scale CAR-NK cells mainly rely on mRNA transfection and viral vector transduction. However, mRNA CAR-NK cells were not stable in CAR expression while viral vector transduction mostly ended up with low efficiency. In this chapter, we described an optimized protocol to generate CAR-NK cells by using the piggyBac transposon system via electroporation and to further expand these engineered CAR-NK cells in a large scale together with artificial antigen-presenting feeder cells. This method can stably engineer human primary NK cells with high efficiency and supply sufficient scale of engineered CAR-NK cells for the future possible clinical applications.

A CD123-specific chimeric antigen receptor augments anti-acute myeloid leukemia activity of Vγ9Vδ2 T cells.

Aim: To investigate whether anti-CD123 chimeric antigen receptor (CAR)-expressing Vγ9Vδ2 T cells could be an alternative for acute myeloid leukemia (AML) treatment. Materials & methods:Ex vivo expanded Vγ9Vδ2 T cells were electroporated with anti-CD123 CAR-encoding mRNA. The effector function and specificity of the modified Vγ9Vδ2 T cells were examined by in vitro cytotoxicity, degranulation and cytokine release level. The in vivo function was analyzed using the xenograft KG1-luc model with NOD-SCID-γc-/- mice. Results: The modified Vγ9Vδ2 T cells exhibited significantly improved effector activities against both AML cell lines and primary AML cells in vitro. In the xenograft mouse model, the modified Vγ9Vδ2 cells displayed an enhanced tumor control potency. Conclusion: Anti-CD123 CAR-expressing Vγ9Vδ2 T cells may serve as an alternative way to target AML.

piggyBac system to co-express NKG2D CAR and IL-15 to augment the in vivo persistence and anti-AML activity of human peripheral blood NK cells.

Promising progress has been made in adoptive transfer of allogeneic natural killer (NK) cells to treat relapsed or refractory acute myeloid leukemia (AML). In this regard, chimeric antigen receptor (CAR)-modification of NK cells is considered as a compelling approach to augment the specificity and cytotoxicity of NK cells against AML. Using a non-viral piggyBac transposon technology and human peripheral blood-derived primary NK cells, we generated CAR-NK cells to target NKG2D ligands and demonstrated their in vitro activity in lysing cancer cells expressing the ligands and in vivo efficacy in inhibiting tumor growth in a xenograft KG-1 AML model. We further generated CAR-NK cells co-expressing transgenes for the NKG2D CAR and interleukin-15 (IL-15). The ectopic expression of IL-15 improved the in vitro and in vivo persistence of NKG2D CAR-NK cells, leading to enhanced in vivo tumor control and significant prolongation of mouse survival in the KG-1 AML model. Collectively, our findings demonstrate the ectopic expression of IL-15 as an important means to improve the antileukemic activity of NKG2D CAR-NK cells. Our study further illustrates the feasibility of using the piggyBac non-viral platform as an efficient and cost-effective way for CAR-NK cell manufacturing.

Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells.

BACKGROUND: Redirection of natural killer (NK) cells with chimeric antigen receptors (CAR) is attractive in developing off-the-shelf CAR therapeutics for cancer treatment. However, the site-specific integration of a CAR gene into NK cells remains challenging. METHODS: In the present study, we genetically modified human induced pluripotent stem cells (iPSCs) with a zinc finger nuclease (ZFN) technology to introduce a cDNA encoding an anti-EpCAM CAR into the adeno-associated virus integration site 1, a "safe harbour" for transgene insertion into human genome, and next differentiated the modified iPSCs into CAR-expressing iNK cells. RESULTS: We detected the targeted integration in 4 out of 5 selected iPSC clones, 3 of which were biallelically modified. Southern blotting analysis revealed no random integration events. iNK cells were successfully derived from the modified iPSCs with a 47-day protocol, which were morphologically similar to peripheral blood NK cells, displayed NK phenotype (CD56+CD3-), and expressed NK receptors. The CAR expression of the iPSC-derived NK cells was confirmed with RT-PCR and flow cytometry analysis. In vitro cytotoxicity assay further confirmed their lytic activity against NK cell-resistant, EpCAM-positive cancer cells, but not to EpCAM-positive normal cells, demonstrating the retained tolerability of the CAR-iNK cells towards normal cells. CONCLUSION: Looking ahead, the modified iPSCs generated in the current study hold a great potential as a practically unlimited source to generate anti-EpCAM CAR iNK cells.

Generation of Mesenchymal Stromal Cells with Low Immunogenicity from Human PBMC-Derived ß2 Microglobulin Knockout Induced Pluripotent Stem Cells.

Mesenchymal stromal cells (MSCs) are viewed as immune-privileged cells and have been broadly applied in allogeneic adoptive cell transfer for regenerative medicine or immune-suppressing purpose. However, the surface expression of human leukocyte antigen (HLA) class I molecules on MSCs could still possibly induce the rejection of allogeneic MSCs from the recipients. Here, we disrupted the ß2 microglobulin (B2M) gene in human peripheral blood mononuclear cell-derived induced pluripotent stem cells (iPSCs) with two clustered regulatory interspaced short palindromic repeat (CRISPR)-associated Cas9 endonuclease-based methods. The B2M knockout iPSCs did not express HLA class I molecules but maintained their pluripotency and genome stability. Subsequently, MSCs were derived from the HLA-negative iPSCs (iMSCs). We demonstrated that B2M knockout did not affect iMSC phenotype, multipotency, and immune suppressive characteristics and, most importantly, reduced iMSC immunogenicity to allogeneic peripheral blood mononuclear cells. Thus, B2M knockout iPSCs could serve as unlimited off-the-shelf cell resources in adoptive cell transfer, while the derived iMSCs hold great potential as universal grafts in allogeneic MSC transplantation.

Beta-2 microglobulin knockout K562 cell-based artificial antigen presenting cells for ex vivo expansion of T lymphocytes.

Aim: The human K562 leukemia cell line as a scaffold of artificial antigen presenting cells (aAPCs) for ex vivo lymphocyte expansion does not usually express major histocompatibility complex (MHC) molecules. However, when stimulated by supernatants from human T lymphocyte cultures, K562 cells upregulate ß-2 microglobulin (B2M) and MHC class I expression, which would induce allo-specific T cells. Methods: We disrupted the B2M locus in K562 cells by CRISPR/Cas9 and achieved MHC class I-negative K562 cells. Results: We further generated K562-based MHC class I-negative aAPC line by zinc-finger nuclease mediated insertion of costimulatory molecules into the AAVS1 locus. This aAPC line could attenuate allogeneic immune responses but support robust antigen-independent and CD19 antigen-specific chimeric antigen receptor-T cell expansion in vitro. Conclusion: B2M-knockout K562 cells provide a new scaffold for aAPC construction and broader application in adoptive immunotherapies.

Large-scale expansion of Vγ9Vδ2 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor-modified effector cells.

Vγ9Vδ2 T cells are a minor subset of lymphocytes in the peripheral blood that has been extensively investigated for their tolerability, safety and anticancer efficacy. A hindrance to the broad application of these cells for adoptive cellular immunotherapy has been attaining clinically appropriate numbers of Vγ9Vδ2 T cells. Furthermore, Vγ9Vδ2 T cells exist at low frequencies among cancer patients. We, therefore, sought to conceive an economical method that allows for a quick and robust large-scale expansion of Vγ9Vδ2 T cells. A two-step protocol was developed, in which peripheral blood mononuclear cells (PBMCs) from healthy donors or cancer patients were activated with Zometa and interleukin (IL)-2, followed by co-culturing with gamma-irradiated, CD64-, CD86- and CD137L-expressing K562 artificial antigen-presenting cells (aAPCs) in the presence of the anti-CD3 antibody OKT3. We optimized the co-culture ratio of K562 aAPCs to immune cells, and migrated this method to a G-Rex cell growth platform to derive clinically relevant cell numbers in a Good Manufacturing Practice (GMP)-compliant manner. We further include a depletion step to selectively remove αß T lymphocytes. The method exhibited high expansion folds and a specific enrichment of Vγ9Vδ2 T cells. Expanded Vγ9Vδ2 T cells displayed an effector memory phenotype with a concomitant down-regulated expression of inhibitory immune checkpoint receptors. Finally, we ascertained the cytotoxic activity of these expanded cells by using nonmodified and chimeric antigen receptor (CAR)-engrafted Vγ9Vδ2 T cells against a panel of solid tumor cells. Overall, we report an efficient approach to generate highly functional Vγ9Vδ2 T cells in massive numbers suitable for clinical application in an allogeneic setting.

Chimeric switch receptor: switching for improved adoptive T-cell therapy against cancers.

Adoptive T-lymphocyte transfer-based immunotherapy for cancers has seen huge leaps with both CARs and engineered TCRs. Despite this, issues relating to safety and efficacy persist. To address this, chimeric switch receptors have been created to reverse the outcomes of their original signaling pathways in order to confer immune cells with the ability to overcome the immunosuppressive tumor microenvironment and to allow them to have greater in vivo persistence. Activating switch receptors exploit the inhibitory molecules expressed by cancer cells to further stimulate the tumor antigen-specific T lymphocytes. On the other hand, inhibitory switch receptors inhibit the effects of tumor-reactive T lymphocytes on unintended targets. This paper reviews the switch receptors reported thus far, and lists out potential improvements and future works.

Maternal Supply of Cas9 to Zygotes Facilitates the Efficient Generation of Site-Specific Mutant Mouse Models.

Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply. We have investigated the feasibility of supplying Cas9 genetically in more detail and for this purpose we report the generation of a transgenic mice which overexpress Cas9 ubiquitously, via a CAG-Cas9 transgene targeted to the Gt(ROSA26)Sor locus. We show that zygotes prepared from female mice harbouring this transgene are sufficiently loaded with maternally contributed Cas9 for efficient production of embryos and mice harbouring indel, genomic deletion and knock-in alleles by microinjection of guide RNAs and templates alone. We compare the mutagenesis rates and efficacy of mutagenesis using this genetic supply with exogenous Cas9 supply by either mRNA or protein microinjection. In general, we report increased generation rates of knock-in alleles and show that the levels of mutagenesis at certain genome target sites are significantly higher and more consistent when Cas9 is supplied genetically relative to exogenous supply.

Designed transcription activator-like effector proteins efficiently induced the expression of latent HIV-1 in latently infected cells.

HIV latency is the foremost barrier to clearing HIV infection from patients. Reactivation of latent HIV-1 represents a promising strategy to deplete these viral reservoirs. Here, we report a novel approach to reactivate latent HIV-1 provirus using artificially designed transcription activator-like effector (TALE) fusion proteins containing a DNA-binding domain specifically targeting the HIV-1 promoter and the herpes simplex virus-based transcriptional activator VP64 domain. We engineered four TALE genes (TALE1-4) encoding TALE proteins, each specifically targeting different 20-bp DNA sequences within the HIV-1 promoter, and we constructed four TALE-VP64 expression vectors corresponding to TALE1-4. We found that TALE1-VP64 effectively reactivated HIV-1 gene expression in latently infected C11 and A10.6 cells. We further confirmed that TALE1-VP64 reactivated latent HIV-1 via specific binding to the HIV-LTR promoter. Moreover, we also found that TALE1-VP64 did not affect cell proliferation or cell cycle distribution. Taken together, our data demonstrated that TALE1-VP64 can specifically and effectively reactivate latent HIV-1 transcription, suggesting that this strategy may provide a novel approach for anti-HIV-1 latency therapy in the future.